1. Field of the Invention
The present invention relates to a technology for efficiently transferring a gene into a eukaryotic algal cell with cell-wall, by performing electroporation through application of multiple square-wave pulses in three steps with total electric energy of a first electric pulse adjusted within a predetermined range.
2. Description of the Related Art
Technical problem in gene transfer into eukaryotic algae
In recent years, novel and useful species of eukaryotic algae have been discovered one after another, and utilization of microalgae that produce oils and fats through photosynthesis is expected to be a key to next-generation biofuel production. Accordingly, in order to produce strains of those algae having useful traits, there has been a demand for development of gene transfer and transformation technologies.
However, cells of the eukaryotic algae have thick cell walls for protecting the cells as with general plant cells, and hence significant technical problems exist in performing gene transfer and transformation.
As a current transformation technology for eukaryotic algal cells, specifically, there may be given (1) a ‘glass bead method,’ which involves allowing algal cells, DNA to be transferred, and glass beads to coexist in a solution, and agitating the solution vigorously for a certain period of time, thereby causing damage to the cells to transfer the DNA into the cells (see Kindle, K.: High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA, 87, p 1228-1232 (1990)). In addition, (2) an ‘electroporation method’ has also been performed, which involves allowing algal cells and DNA to be transferred to coexist in a solution, and discharging a current charged in a capacitor, thereby causing damage to the cells to transfer the DNA into the cells (see Shimogawara, K., Fujiwara, S., Grossman, A., and Usuda, H.: High-efficiency transformation of Chlamydomonas reinhardtii by electroporation. Genetics, 148, p 1821-1828 (1998)).
However, in each of the glass bead method and the electroporation method, it is necessary to perform the gene transfer under a state in which the target algal cells have been converted into “protoplasts,” i.e., the cell walls have been removed from the cells. Accordingly, when any of those methods is performed, it is necessary to establish systems for the conversion into protoplasts and their regeneration depending on the species of the target alga. (It should be noted that, when the protoplasts are not prepared, it is necessary to produce a cell wall-less special mutant strain.)
Therefore, it is recognized to be difficult in reality to use any of those methods for most species of algae other than certain algae (e.g., Chlamydomonas reinhardtii). In addition, the following problem has been pointed out. That is, when any of those methods is performed for Chlamydomonas reinhardtii or the like, the conversion into protoplasts and their regeneration are time-consuming processes, and hence reproducibility is low depending on experimenters.
In addition, as a method that allows the preparation of protoplasts to be omitted, there is given (3) a method involving using a ‘gene gun (particle gun).’ The method is a method involving preparing gold particles or tungsten particles coated with DNA to be transferred, and shooting the particles into cells with gunpowder or a nitrogen gun to transfer the DNA into the cells (see Boynton, J., Gillham, N., Harris, E., Hosler, J., Johnson, A., Jones, A., Randolph-Anderson, B., Robertson, D., Klein, T., Shark, K., and Sanford, J.: Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science, 240, p 1534-1538 (1998), and Blowers, A., Bogorad, L., Shark, K., and Sanford, J.: Studies on Chlamydomonas chloroplast transformation: foreign DNA can be stably maintained in the chromosome. Plant Cell, 1, p 123-132 (1989)).
However, the method needs an expensive and special device and costly consumables, and requires a long period of time for sample preparation. Hence, the method is recognized to be difficult to utilize with general laboratory-level equipment. There is also a problem in that the method is not suitable for performing high-throughput or large-scale treatment. Further, the following problem has also been pointed out. That is, sufficient transformation efficiency cannot be expected because of its low gene transfer frequency.
Necessity of Gene Transfer Technology for Eukaryotic Algae
As described above, technical problems exist in transferring an exogenous gene into eukaryotic algal cells, and gene transfer into species of algae other than certain species such as Chlamydomonas reinhardtii is substantially difficult in the present situation. Accordingly, there has been desired development of a highly general-purpose gene transfer method that is applicable to any species of algae. In particular, there is a demand for development of a technology that is readily applicable to various useful algae such as green algae and diatoms.
In addition, also in the case of performing gene transfer into Chlamydomonas reinhardtii or the like, it is necessary to prepare gametolysin, which is a cell wall lytic enzyme, and hence there has been desired development of a method that enables efficient gene transfer within a shorter period of time.